Use of enzymatic additives, in particular proteolytic enzymes, in detergent compositions to enable removal of protein based soilings has been amply documented. See for example the published European Patent Applications EP-A-0220921 and EP-A-0232269, U.S. Pat. Nos. 4,480,037 and Re 30,602, and the article "Production of Microbial Enzymes", Microbial Technology, vol. 1 (1979) 281-311, Academic Press.
Detergent compositions, which are applied for hard surface cleaning, toilet cleaning, dish washing and laundry cleaning, may be in a powder, liquid or paste form. Laundry detergents are generally divided into two major types, liquids and powders.
Proteolytic enzymes are generally difficult to combine with detergent compositions. They must be stable and active during application, for example in removing proteinaceous stains from textile during washing at temperatures ranging from about 10.degree. C. to over 60.degree. C. Furthermore they must be stable for prolonged periods of time during storage in the detergent product. Consequently, enzymes have to be stable and functional in the presence of sequestering agents, surfactants, high alkalinity, often bleaching agents, and elevated temperature. As there exist neither universal laundry detergents nor universal washing conditions (pH, temperature, sud-concentration, water hardness) that are used all over the world, the demands on enzymes may vary based on the type of detergent in which they are used and on the washing conditions.
A commercially important group of proteases is that of the so-called high alkaline proteases, derived from alkalophilic Bacilli. The commercially available high alkaline protease product MAXACAL.RTM. (Gist-brocades/IBIS) contains the serine protease "PB92", derived from Bacillus novo sp. PB92 (see U.S. Pat. Re. No. 30,602). Its amino acid sequence is disclosed in EP-A-0283075 and EP-A-0284126. Also SAVINASE.RTM. (Novo-Nordisk) is a member of this group. SAVINASE contains the "Subtilisin 309" enzyme, which is derived from Bacillus strain NCIB 10147 (U.S. Pat. No. 3,723,750). Its amino acid sequence is disclosed in WO 89/06279, where the strain is referred to as Bacillus lentus. The amino acid sequences of these two proteases appear to differ only at position 85 (taking the residue numbering of the PB92 protease, which corresponds to position 87 in the BPN' numbering), where PB92 has an asparagine ("N") in the one letter amino acid code) and "Subtilisin 309" a serine ("S").
Since the PB92 protease is active in stain removing at alkaline pH-values, it is commonly used as a detergent additive, together with detergent ingredients such as surfactants, builders and oxidizing agents. The latter agents are mostly used in powder form. The detergent additive may also contain other enzymes, for example amylases, cellulases and/or lipases, as far as they are compatible with the protease. PB92 protease has a high stain removing efficiency as compared to other proteases, such as the "classic" subtilisins which are well known in the art. This means that less PB92 protease is needed to obtain the same wash performance. Sensitivity to oxidation is an important drawback of the PB92 protease and all other known serine proteases used for application in detergents.
Originally the commercially available alkaline proteases such as MAXACAL.RTM. were developed for application in detergents at enhanced temperatures in the range 40-60.degree. C. However nowadays, because the growing emphasis on ecomomy, there is an ongoing tendency to switch to lower temperatures. As a consequence the lower wash performance at reduced temperatures, e.g. 15-25.degree. C., is an important handicap of the excisting commercially alkaline proteases.
There are several ways of obtaining new enzymes for an intended application, which are all known to the skilled artisan. Modification of existing enzymes by protein engineering is likely to be the most popular and effective method nowadays.
The most specific way of obtaining modified enzymes is by site-directed mutagenesis, enabling specific substitution of one or more amino acids by any other desired amino acid. EP-A-0130756 exemplifies the use of this technique for generating mutant protease genes which can be expressed to give modified proteolytic enzymes. A very effective method is the oligonucleotide mediated site-directed mutagenesis, which allows a number of different mutations to be introduced at a specific part of a DNA sequence by using a single synthetic oligonucleotide preparation.
For a comprehensive summary of the various detergent compositions and enzymes, their physical forms, the conditions which the enzymes have to meet for optimal functioning, the problems and limitations of the currently available enzymes for use in detergent enzyme compositions, preparation and screening of mutant proteases, etc., reference may be made to EP-A-0328229, which is incorporated herein by reference.
WO 89/06279 claims inter alia mutants of the "Subtilisin 309" protease, in which one or more residues at the following positions are substituted (taking the original BPN' residue numbering): 6, 9, 11-12, 19, 25, 36-38, 53-59, 67, 71, 89, 104, 111, 115, 120, 121-122, 124, 128, 131, 140, 153-166, 168, 169-170, 172, 175, 180, 182, 186, 187, 191, 194, 195, 199, 218, 219, 222, 226, 234-238, 241, 260-262, 265, 268, or 275. The, number of examples in this reference describing mutants which have been actually made and tested is restricted to only eight, while no more than four positions are involved. These mutants are: S153A, G195D, G195E, N218S, [G195E M222A], [G195E M222C], M222A, and M222C.
EPA-A-0328229 discloses and claims inter alia mutant proteases which have at least 70% homology with the amino acid sequence of PB92 serine protease and differ by at least one amino acid residue at a selected site corresponding to 32, 33, 48-54, 58-62, 94-107, 116-118, 123-134, 150, 152-156, 158-161, 164, 166, 169, 175-186, 197, 198 and 203-216, 235, 243 and 259 in said PB92 serine protease, and having improved wash performance and/or improved stability relative to said PB92 serine protease. This reference is exemplified by 69 mutants, in which 17 positions are involved.
Despite the progress which seems to have been made in the past few years, there is a continuing interest in the development of new proteolytic enzymes with improved properties which make them more attractive for use in detergents. These properties may include, but are not limited to, better wash performance, improved stain removing ability at low laundering temperature, improved stability upon storage, or improved stability while they are used.